A candidate reference measurement procedure for the quantification of α-synuclein in cerebrospinal fluid using an SI traceable primary calibrator and multiple reaction monitoring

Abstract

α-synuclein aggregation is an important hallmark of neurodegenerative diseases such as Parkinson's disease (PD) and Lewy body dementia. α-synuclein has been increasingly used as a diagnostic biomarker in PD and other synucleinopathies. Current clinical assays rely on antibody-based immunoassays to detect α-synuclein, which possess high sensitivity, afford high throughput and require small sample volumes. The utility of these assays, however, may be compounded by the specificity, selectivity and batch-to-batch heterogeneity of the antibody used, which can lead to deviations in the total amount of the protein measured when comparing results among different laboratories. Similarly, current mass spectrometry-based quantification methods for α-synuclein lack well-defined, value assigned calibrators to ensure comparability of measurements. Therefore, there is still an unmet need for the standardisation of clinical measurements for α-synuclein that can be achieved by the development of reference measurement procedures (RMPs) utilising calibrators traceable to the SI (International System of Units). Here, we report a candidate RMP for α-synuclein, using an SI traceable primary calibrator and an isotope dilution mass spectrometry (IDMS) approach to address this need. The gravimetrically prepared primary calibrator was traceably quantified utilising a combination of amino acid analysis (AAA) and quantitative nuclear magnetic resonance (qNMR) for value assignment. An optimised targeted sample clean-up procedure involving a non-denaturing Lys-C digestion and solid-phase extraction strategy was devised, followed by the development of a targeted multiple reaction monitoring (MRM) method for the quantification of α-synuclein in cerebrospinal fluid (CSF). This candidate RMP was then deployed for the sensitive detection and accurate quantification of multiple proteotypic α-synuclein peptides in patient derived CSF samples. The LC-MS based results were subsequently compared to immunoassay data to assess the overall performance of our approach. The development and adoption of this candidate RMP, along with the availability of the SI traceable primary calibrator will allow for reliable quantifications of α-synuclein in CSF by an LC-MS based assay. The RMP will potentially contribute towards the standardisation of this important biomarker and may lead to future interlaboratory comparisons.